This invention relates to detection of soluble factor secretion by activated T-cells. In particular, this invention relates to modifications of the standard ELISPOT assay.
Therapeutic strategies ranging from vaccine design to T cell specific immunosuppression require identification of immunodominant T cell epitopes and enumeration of T cell frequency. Several assays are currently employed to provide this information. Modified proliferation assays have been used to identify T cell epitopes based on stimulation indices of .gtoreq.2.0 (Plebanski, M., and Burtles, S. S., J. Immunol. Meth. 170:15 (1994)), but this assay is extremely sensitive to variations in serum and often proves difficult for large scale clinical screenings. The limiting dilution assay (LDA) employs relatively large PBMC quantities and two rounds of in vitro stimulation to detect the T cell response to whole antigens or peptides (Sharrock, C. E. M. et al., Immunol. Today 11:281-286 (1990)). This assay has provided estimates of antigen specific CD4+ T cell frequencies ranging from approximately 1/10.sup.3 -1/10.sup.5 for alloreactive T cells (Sharrock supra) to 10.sup.6 -1/10.sup.7 for autoreactive T cells (Weiner, H. L. et al., Science 259:1321 (1993)). The LDA has been used to monitor efficacy in clinical trials, but the quantities of PBMC's (peripheral blood mononuclear cells) required limit the application of this assay in cases requiring frequent blood draws or the screening of large numbers of candidate peptides. Several flow cytometric methods can detect T cell activation by upregulation of characteristic markers such as CD69. Activation-induced T cell lymphokine production can be measured by flow cytometry using a monensin block of secretion, saponin permeabilization, and indirect immunofluorescent staining (Jung, T. et al., J. Immunol. Meth. 159:197 (1993)), or by trapping of secreted lymphokines on the surface of the secreting cell (Manz, R. et al., Proc. Natl. Acad. Sci. USA 92:1921 (1995)). These flow cytometry techniques are sufficiently sensitive when a relatively high frequency of T cells respond, as occurs in alloreactivity or superantigen stimulation, but they cannot detect most rare antigen-specific T cells. ELISA assays of lymphokine secretion are similarly limited to cases in which the responses of primed T cells, T cell clones, or high frequency T cells are measured. In situ hybridization of lymphokine mRNA is sufficiently sensitive to detect antigen-specific T cells with frequencies in the range of 1/10.sup.4 -1/10.sup.5 (Link, J. et al., Neurol. 44:728 (1994); Link, J. et al., Ann. Neurol. 35:197 (1994)), but this technique is not readily scalable to large sample numbers.
A modification of the ELISA assay (enzyme-linked immunosorbent assay), termed the immunospot or ELISPOT assay, has been developed to detect lymphokine secretion by individual T cells following antigen stimulation (Czerinsky, C., et al., J. Immunol. Methods 110:29-36 (1988); Olsson, T. et al., J. Clin. Invest. 86:981-985 (1990)). However, the sensitivity of the standard ELISPOT assay is low. For example, for many multiple sclerosis (MS) patients, the standard ELISPOT assay of T cell responses to autoantigens can only be detected in cells sampled from the CSF, which entails difficult sampling and low cell yield. Identifying peptide epitopes within autoantigens such as MBP (myelin basic protein) by this assay is even more difficult given the relatively low precursor frequency. Furthermore, counting ELISPOT sample wells under light microscopy is slow and somewhat subjective. It would be desirable to have improved methods of measuring lymphokine secretion by activated T-cells, particularly those which occur at low frequency. This invention fulfills this and related needs.